The proposed research is part of program that seeks to determine why people with cystic fibrosis (CF) have chronic airway infections. The general hypothesis is that defective airway submucosal glands produce mucus that is under-hydrated, compromising mucus clearance and the bio-availability of antimicrobial and anti-inflammatory compounds. In previous work we showed a profound defect in the response of CF glands to agents that elevate cAMP. To progress from those observations we propose 4 specific aims. Aim 1: To determine how local (intrinsic) neurons, especially those containing VIP, control gland secretion directly and via interaction with cholinergic input. Aim 2: To study secretory mechanisms at the single cell level within four anatomical compartments of the glands: the serous acini, mucous tubules, collecting duct and ciliated duct. Cellular responses are quantified using differential interference contrast (DIC) time-lapse digital imaging with which we optically section isolated, functioning glands from control and CF subjects. We will study how each of the four compartments respond to agents that increase [cAMP]i, [Ca2+]i or both, in the presence of control solution (Krebs-bicarbonate buffer) or solutions in which ion substitutions or transport inhibitors are used to dissect the transport processes within the glands. We will also clarify mechanisms for fluid and protein secretion by serous and mucous cells: our imaging methods allow us to quantify individual exocytotic events. Aim 3: To determine the pH of luminal mucus within the 4 gland compartments by using ratiometric imaging of fluorescent indicators (BCECF) injected into the gland lumen. Aim 4: To patch clamp partially dissociated gland serous and mucous cells, to obtain evidence for against the hypothesis that CFTR in the only apical anion channel in serous cells and is absent from mucous cells. Successful completion of these aims will clarify the contribution of glands, which produce approximately 95% of airway mucus, to the mucus clearance and chemical shield components of airway innate defenses.